PROJECT SUMMARY/ABSTRACT A critical property of all eukaryotic cells is the proper distribution of organelles. The arrangement of organelles is distinct for each cell-type. Moreover, as part of differentiation, organelles are moved to new locations as a cell acquires new functions. There are large gaps in current knowledge of the mechanisms that move organelles to the proper place at the correct time. To address these gaps, this proposal is focused on the inheritance of the vacuole/lysosome. Vacuole/lysosome inheritance in yeast provides an excellent model as it involves cell cycle-dependent as well as spatial regulation of a myosin V molecular motor (MyoV). Defects in MyoV transport underlie some human diseases, including neurological diseases, and some fatal disorders of the gastrointestinal tract. Importantly human and yeast MyoV are highly similar. A major mode of MyoV regulation is via control of its attachment to and detachment from the vacuole cargo during cell cycle progression. Given that little is known about how any type of molecular motor attaches or detaches from cargoes, the proposed studies will address a critical gap in knowledge. Aim 1 will determine how a MyoV cargo is released from the motor. Preliminary data suggest that the release occurs via step-wise post- translational modification of an adaptor protein that links MyoV to its cargo. These steps are highly regulated. Some candidate factors required for the regulation have been uncovered. A combination of genetic and biochemical analyses will be used to determine the specific functions of these factors. Studies of vacuole inheritance also led to the unexpected discovery that the vacuole/lysosome is essential for cell-cycle progression, and acts in parallel with the cyclin-dependent kinase pathway. Moreover, lysosomes may have a similar role in the mammalian cell-cycle. Insights into how organelles regulate the cell- cycle may provide new approaches to treat diseases such as cancer, where normal control of the cell-cycle is lost. Characterization of a role for the lysosome in cell-cycle progression, will likely uncover new targets for cancer that have not been previously explored. Aim 2 seeks to gain mechanistic insights into how the vacuole contributes to the cell-cycle. A portion of the Aim is based on a new discovery that following stress, a previously unidentified pathway re-initiates a paused cell-cycle. Yeast genetics, chemical inhibition and in vitro assays will be used to map the specific pathways required for the re-initiation of the cell cycle. Excitingly, proteins that we already identified in the yeast pathway are conserved in mammalian cells. Thus, in addition, this proposal will test whether the mammalian lysosome plays an essential role in cell-cycle progression.